The present invention relates to hybridomas producing a monoclonal antibody reactive to human cholesterol ester transfer protein(CETP), a monoclonal antibody and its fragment reactive to human CETP, an immobilized monoclonal antibody and immobilized antibody fragment, a labeled monoclonal antibody and labeled antibody fragment, a kit for detection, assay, separation or purification of human CETP, a method for detection, assay, separation and purification of human CETP and a pharmaceutical composition containing said monoclonal antibody or said antibody fragment.
There are three types of cholesterol, free type, long chain fatty acid type and ester type, in all the tissues and blood plasma in organisms. The former two play important roles in the composition of cell membranes. The latter is physiologically inactive and exists mainly in a storage form. Cholesterols in a body are derived from ingestion in the small intestine or from biosynthesis in various tissues, especially in the liver. Most cholesterols are derived from biosynthesis in the liver.
Free cholesterol biosynthesized and secreted from the liver is incorporated in very low density lipoprotein(VLDL). Then, by the actions of lipoprotein lipase(LPL) and hepatic triglyceride lipase(HTGL), it is metabolized to low density lipoprotein (LDL) through intermediate density lipoprotein (IDL). By the incorporation of LDL into LDL receptors of peripheral cells, free cholesterol is supplied to cells.
There is a pathway from the peripheral cells to the liver called the cholesterol reverse transfer system, which goes in reverse of the way from liver to peripheral cells as mentioned above. Surplus free cholesterol supplied in the peripheral cells from the liver is drawn by high density lipoprotein(HDL) in blood. Then, by the action of lecithin cholesterol acyl transferase(LCAT), it is converted to cholesterol ester and is stored in high density lipoprotein(HDL) in blood. By the action of cholesterol ester transfer protein(CETP), the cholesterol ester stored in HDL is transferred to VLDL, IDL or LDL in blood. By the incorporation of VLDL, IDL or LDL cholesterol ester received through LDL receptors in the liver, cholesterol is indirectly transferred to the liver.
Recently, the reverse cholesterol transfer system has drawn much attention as a mechanism for preventing the peripheral cells from accumulating cholesterol and thereby preventing atherosclerosis. In fact, as for HDL which plays an important role in the reverse cholesterol transfer system, many epidemiological surveys show that decrease of cholesterol ester in blood HDL is one of the risk factors of coronary artery disorders. It is now well recognized that HDL is a lipoprotein having anti-arteriosclerosis action.
In addition to the importance of blood HDL, it became recognized that CETP is also important because it mediates transfer of cholesterol ester in HDL into blood LDL. Therefore, it became an urgent matter to elucidate the relationship between CETP and various diseases such as CETP deficiency, hyperlipidemia, hyperalphalipoproteinemia, hypercholesterolemia, hypolipidemia, arteriosclerosis, diabetes and nephrotic syndrome.
For instance, it is experimentally demonstrated that several times higher CETP are secreted in blood of the patients with hyperlipidemia compared to those of healthy volunteers. The following findings have been made in relation to arteriosclerosis. When CETP activity is low, arteriosclerosis is not easily induced and the level of HDL cholesterol is high. In contrast, when CETP activity is high, arteriosclerosis is easily induced and the level of HDL cholesterol is low. Such relationships have been experimentally demonstrated(Current Therapy, vol. 7, 9:36-45(1989)).
In order to elucidate the relationship between various diseases and CETP, assay methods for CETP in body fluids such as blood plasma from a healthy person or patient having the above various diseases, especially immunoassay methods such as radio-immunoassay(RIA) by using a monoclonal antibody against CETP(anti-CETP monoclonal antibody), or enzyme-immunoassay(EIA, ELISA) are being developed together with an anti-CETP monoclonal antibody used for the assay methods.
As to EIA(ELISA) assay with the anti-CETP monoclonal antibody, there are the examples by Imai et al.(Japanese Unexamined Patent publication No.HEI6-169793), Nakano, et al.,(Arteriosclerosis, vol. 19, 11:951, No. 22, (1991)), Takahama, et al.(Arteriosclerosis, vol. 20, 10:837, No. 135, (1992)), Sato, et al.(Arteriosclerosis, vol. 20, 10:836, No. 134, (1992)), H. Mezdur, et al.(Clinical Chemistry, vol. 40, 4:593-597(1994)). Clark et al.(Journal of Lipid Research, vol. 36,:876-889(1995)) reported the sandwich ELISA method by using two kinds of anti-CETP monoclonal antibodies or their Fabxe2x80x2 fragments.
However, these assay methods require complicated handling. Before assay, a plasma sample has to be heat-treated at 95-100xc2x0 C. and/or pre-treated with surface active agents (surfactants, detergent) such as TWEEN 20 and TRITON X-100. Since heat treatment of a plasma sample causes denaturation of CETP in the sample, the assay result tells only the amount of denatured CETP. Therefore, it is impossible to assay intact CETP in a plasma sample accurately.
Also, R. Clark et al.(FASEB Journal, vol. 8, 7:A1343 No. 495(1994)), Takahashi et al.(Arteriosclerosis, vol. 20, 10:837 No. 136(1992) and ibid. vol. 21, 3:209 No. 97(1993)), Kanamitu et al.(ibid. vol. 21, 3:209 No. 98(1993)) and Waki et al.(ibid. vol. 22, 5:441 No. 194(1994)) reported assay results on CETP in plasma samples by ELISA, however, they did not describe in detail their preparation methods, properties of anti-CETP antibodies and specific assay procedures.
Regarding RIA assay methods with anti-CETP monoclonal antibody, Fukazawa et al.(Study on lipid biochemistry, vol. 34:163-166(1992)), Y. Marcel et al.(J. Clin. Invest., vol. 85:10-17(1990) and Adv. Exp. Med. Biol., vol. 243:225-230(1988)), Fukazawa et al.(J. Biochem., vol. 111:696-698(1992)), J.Koizumi et al. (Atherosclerosis, 90:189-196(1991), M. Brown et al.(Nature, vol. 342:448-451(1989)) and V. Dangremont et al.(Clinica Chimica Acta, vol. 231:147-160(1994)) published reports thereon.
However, these RIA methods also had the same defects as the EIA(ELISA) method had. The defects were complexity of handling (the methods required pre-treatment of a plasma sample with a detergent) and/or insufficient sensitivity for detection.
As described above, many researchers tried to establish an assay method for CETP. However, there is no report of a convenient and sensitive system which needs no pretreatment of a sample and which determines conveniently the amount of intact CETP with high sensitivity.
To elucidate the relationship between the diseases (such as arteriosclerosis, hyperlipidemia hyperalphalipoproteinmia and hypercholesterolemia) and CETP that plays an important role in the cholesterol reverse transfer system which may be highly related to the onset of the diseases, there is a strong reason for developing a simple and sensitive assay method that can be widely applied to clinical purposes to assay intact CETP in a body fluid including blood plasma from healthy persons or any patients, and for developing a monoclonal antibody useful for said assay method. However, these have not yet been established. The present invention provides an assay method widely applicable for clinical purposes and an anti-human CETP monoclonal antibody which is very useful not only for said assay method but also as a reagent for separation and purification of CETP and as a pharmaceutical product.
The inventors of the present invention extensively investigated assay methods for CETP in a human body fluid that can be widely applicable for clinical purposes and monoclonal antibodies against human CETP used for said assay methods. By using biologically active purified human CETP as an immunogen, the inventors succeeded in preparing three anti-human CETP monoclonal antibodies which respectively have different high binding specificity(CETP inhibitory activity) to human CETP, especially intact CETP in the human body fluid.
Furthermore, as the three anti-human CETP monoclonal antibodies have high binding specificity(CETP inhibitory activity) to human CETP, especially intact CETP in a human body fluid, the inventors have found that, for assaying intact CETP in the human body fluid(blood plasma etc.), the immunoassay using the anti-human CETP monoclonal antibodies of the present invention can provide a simple and sensitive assay method which has not been previously established.
Particularly, as the three anti-human CETP monoclonal antibodies have high but different binding specificity(CETP inhibitory activity) to human CETP(especially to intact CETP in the human body fluid), by using a combination of any of the two of the three monoclonal antibodies for sandwich ELISA, the inventors were succeeded in developing a simpler and more sensitive assay method widely applicable for clinical purposes.
As the anti-human CETP monoclonal antibodies have high binding specificity(CETP inhibitory activity) to human CETP(especially to intact CETP in the human body fluid), it is possible to assay human CETP simply and sensitively without any pre-treatment such as heat or detergent treatment of a plasma sample using the monoclonal antibodies of the invention.
In addition, as the anti-human CETP monoclonal antibodies of the invention have high CETP inhibitory activity, they are useful as pharmaceuticals for treatment or prevention of various diseases caused by abnormal dynamics of CETP such as arteriosclerosis, hyperlipidemia, high blood HDL and hypercholesterolemia.
The first aspect of the present invention is a monoclonal antibody having at least the following characteristics:
(a) reactive to plasma CETP(Cholesterol Ester Transfer Protein) of a healthy human, hyperlipidemia patients, LCAT(Lecithin Cholesterol Acyl Transferase) deficiency patients or hyper HDL (high density lipoprotein) patients.
(b) not reactive to rabbit CETP at a concentration of 3 xcexcg/ml or below.
(c) not specifically reactive to denatured human CETP.
A second aspect of the present invention is the hybridomas which produce monoclonal antibodies reactive to human CETP, and more specifically, hybridomas #72-1 and #86-2 identified by the Accession Number FERM BP-4944 and FERM BP-4945 respectively.
A third aspect of the present invention is the monoclonal antibodies reactive to human CETP, and more specifically, monoclonal antibodies #72-1 and #86-2, which are derived respectively from the hybridomas #72-1 and #86-2 identified by the Accession Number FERM BP-4944 and FERM BP-4945 respectively.
A fourth aspect of the present invention is a recombinant chimeric monoclonal antibody reactive to human CETP, comprising a variable region derived from that of the above-mentioned monoclonal antibody and a constant region derived from that of human immunoglobulin.
A fifth aspect of the present invention is a recombinant humanized monoclonal antibody reactive to human CETP, in which a part of or the whole of the complementarity determining regions of the hypervariable region is derived from that of the above-mentioned monoclonal antibody, the framework regions of the hypervariable region are derived from that of human immunoglobulin, and the constant region is derived from that of human immunoglobulin.
A sixth aspect of the present invention is an antibody fragment termed F(abxe2x80x2)2 or Fabxe2x80x2 derived from the above-mentioned monoclonal antibody, the above-mentioned recombinant chimeric monoclonal antibody, or the above-mentioned recombinant humanized monoclonal antibody.
The seventh aspect of the present invention is an immobilized monoclonal antibody and an immobilized antibody fragment. The immobilized monoclonal antibody is prepared by immobilizing the monoclonal antibody, the recombinant chimeric monoclonal antibody or the recombinant humanized monoclonal antibody on an insoluble carrier. The immobilized antibody fragment is prepared by immobilizing the above mentioned antibody fragment F(abxe2x80x2)2 or Fabxe2x80x2 on an insoluble carrier.
More specifically, the immobilized monoclonal antibody or the immobilized antibody fragment is that which has been immobilized on a plate, a test tube, a tube, beads, a ball, a filter, a membrane or an insoluble carrier used for affinity column chromatography.
The eighth aspect of the present invention is a labeled monoclonal antibody and a labeled antibody fragment. The labeled monoclonal antibody is prepared by labeling the monoclonal antibody, the recombinant chimeric monoclonal antibody or the recombinant humanized monoclonal antibody with a substance capable of providing detectable signals independently or by reaction with another substance. The labeled antibody fragment is prepared by labeling the antibody fragment, F(abxe2x80x2)2 or Fabxe2x80x2, with a substance capable of providing detectable signals independently or by reaction with another substance.
The monoclonal antibody or antibody fragment can be labeled with an enzyme, a fluorescent material, a chemical luminous material, biotin, avidin or radioisotopes.
A ninth aspect of the present invention is a kit for assay or detection of human CETP comprising at least the monoclonal antibody, the recombinant chimeric monoclonal antibody or recombinant humanized monoclonal antibody, the above-mentioned antibody fragment F(abxe2x80x2)2 or Fabxe2x80x2, the immobilized monoclonal antibody or immobilized antibody fragment, or the labeled monoclonal antibody or the labeled antibody fragment.
Specifically, the invention involves a kit for assay or detection of human CETP comprising the immobilized monoclonal antibody or the immobilized antibody fragment and the labeled monoclonal antibody or the labeled antibody fragment.
Ae tenth aspect of the present invention is a method for assay and detection of human CETP by immunoassay characterized by using at least the monoclonal antibody, the recombinant chimeric monoclonal antibody or the recombinant humanized monoclonal antibody, the antibody fragment F(abxe2x80x2)2 or Fabxe2x80x2, the immobilized monoclonal antibody or the immobilized antibody fragment, or the labeled monoclonal antibody or the labeled antibody fragment.
The first specific aspect is a method for assay or detection of human CETP by immunoassay comprising at least the following steps:
(a)reacting a sample with the above-mentioned immobilized monoclonal antibody or the immobilized antibody fragment; and
(b)reacting the labeled monoclonal antibody or the labeled antibody fragment with an antigen-antibody complex which is formed by binding human CETP in a sample with the immobilized monoclonal antibody or the immobilized antibody fragment.
The second specific aspect is a method for assay or detection of human CETP by immunoassay comprising at least the following steps:
(a) reacting a sample with the above-mentioned labeled monoclonal antibody or the labeled antibody fragment; and
(b) reacting the above-mentioned immobilized monoclonal antibody or the immobilized antibody fragment with an antigen-antibody complex which is formed by binding human CETP in a sample with the labeled monoclonal antibody or the labeled antibody fragment.
The third specific aspect is a method for assay or detection of human CETP by immunoassay comprising at least the following step:
(a) reacting a mixture comprising the immobilized monoclonal antibody or the immobilized antibody fragment, the labeled monoclonal antibody or the labeled antibody fragment and a sample.
The fourth specific aspect is a method for assay or detection of human CETP by immunoassay comprising at least the following step:
(a) reacting a sample and a human CETP standard labeled with a labeling substance capable of providing detectable signals independently or by reaction with another substance with the above-mentioned immobilized monoclonal antibody or the immobilized antibody fragment.
The fifth specific aspect is a method for assay or detection of human CETP by immunoassay comprising at least the following step (a) or following steps (b) and (c):
(a) reacting the above-mentioned monoclonal antibody or antibody fragment with a mixture of a sample and a labeled human CETP standard which is prepared by labeling with a substance capable of providing detectable signals independently or by reacting with other substances;
(b) reacting the above-mentioned monoclonal antibody or the antibody fragment with a sample;
(c) followed by the step (b), reacting the labeled human CETP standard with the reaction mixture of step (b), in which the labeled human CETP standard is prepared by labeling with a substance capable of providing a detectable signal independently or by reacting with other substances.
More specifically, a method for assay or detection of human CETP by immunoassay comprising at least the following steps (a) and (d), or steps (b) to (d):
(a) reacting the monoclonal antibody or the antibody fragment of the present invention with a mixture of a sample and a labeled human CETP standard that is prepared by labeling with a substance capable of providing a detectable signal independently or by reacting with other substances;
(b) reacting the monoclonal antibody or the antibody fragment of the present invention with a sample;
(c) followed by step (b), reacting a labeled human CETP standard with the reaction mixture of step (b), in which the labeled human CETP standard is prepared by labeling with a substance capable of providing a detectable signal independently or by reaction with other substances;
(d) reacting an antiserum derived from a mammal reactive with the monoclonal antibody or the antibody fragment with an antigen-antibody complex which is formed by binding human CETP in the sample or the labeled human CETP standard with the monoclonal antibody or the antibody fragment.
An eleventh aspect of the present invention is a kit for separation or purification of human CETP comprising an immobilized monoclonal antibody or an immobilized antibody fragment.
A twelfth aspect of the present invention is a method for separation or purification of human CETP by affinity chromatography using an immobilized monoclonal antibody or an immobilized antibody fragment. More specifically, it is a purification method for human CETP using column chromatography.
A thirteenth aspect of the present invention is a mouse expressing a DNA encoding human CETP which constantly secretes human CETP the in blood without extrinsic or artificial induction.
A fourteenth aspect of the present invention is a pharmaceutical composition comprising any one of monoclonal antibodies, a recombinant chimeric monoclonal antibody, a recombinant humanized monoclonal antibody or an antibody fragment (F(abxe2x80x2)2 or Fabxe2x80x2), and a pharmaceutically acceptable carrier. More specifically, it is a pharmaceutical composition for treating and/or preventing hyperlipidemia or arteriosclerosis.